Developing an H7N9 flu vaccine is tricky — reliant on both cutting-edge technology and the lowly chicken egg. Every step of the way, things can go wrong.

“The global public health community remains woefully underprepared for an effective vaccine response to a pandemic.”

Michael Osterholm

in an editorial in the Journal of American Medical Association

Battling the lethal H7N9 virus: Inside the lab where vaccine is being developed

In a dark laboratory, Jim Allay flicks on a lamp and holds up a chicken egg against the incandescent light.

The shell glows a smouldering orange and a delicate ribbon of veins is illuminated. Inside, a shadowy figure moves languorously.

“This is the developed embryo,” Allay says, peering at the shadow through safety goggles. He points at a spot on the shell. “We're going to drill our hole right there.”

Allay is a scientist with the St. Jude Children's Research Hospital in Memphis, Tenn., and the chicken embryo he is holding is nine days old.

On the embryo's 10th day of life, Allay will bring it and 159 other fertilized eggs into a high-security lab and inject them with an engineered version of a deadly new virus — an avian flu the World Health Organization says is among the “most lethal” ever seen.

The “unusually dangerous” virus is called H7N9 and it exploded out of the gates in March, causing 131 infections and 36 deaths in fewer than two months. Luckily, H7N9 still lacks the pandemic-stoking ability of spreading easily from human to human — people appear to be catching it from poultry. A new study published Thursday showed the virus has some worrying mutations, and also suggested it could transmit by air, although perhaps not very efficiently. And recently, the virus seems to be slinking back underground, with no new infections since May 8.

But H7N9 is still lurking somewhere and scientists know the longer a virus circulates, the better its chances of mutating into a full-blown pandemic.

This is why on May 10, when the chicken embryos were 10 days old, Allay and his colleagues were injecting the 160 eggs with a vaccine “seed strain” — a virus that contains H7N9 genes but has been stripped of any dangerous properties.

If an H7N9 pandemic erupts, a vaccine made from this seed strain could someday be injected in your arm. But making flu vaccine is a funny business — reliant on both cutting-edge technologies and the primordial processes inside a chicken egg. And every step of the way, there are plenty of opportunities for things to go awry.

St. Jude hospital is one of six WHO collaborating centres for influenza and the only non-government facility in the WHO system developing an H7N9 seed strain. The Toronto Star recently travelled to Memphis to take a peek inside the lab and see what it takes to clear the first of many hurdles in making a pandemic vaccine.

Work on the seed strain began shortly after Dr. Richard Webby — director of St. Jude's WHO collaborating centre and one of the world's leading flu experts — learned of H7N9's debut in an email from Beijing.

“This email is to (notify) you that three novel human cases of avian influenza H7N9 virus were confirmed in China,” the message began. “Two of the three cases are from Shanghai and one from Anhui province.”

The two Shanghai patients, both men, were already dead by the time Webby read the email. The Anhui patient — a 35-year-old housewife who had visited a chicken market one week before getting sick — was still alive but would die nine days later.

But throat swabs had been taken from all three patients and the viruses genetically sequenced. The genomes were then uploaded to an Internet database called GISAID, or Global Initiative on Sharing All Influenza Data.

GISAID was created in 2008, largely in response to growing concerns over H5N1, another anxiety-provoking bird flu that — prior to H7N9 — seemed the prime suspect for igniting the next explosive outbreak. One of GISAID's goals was to encourage rapid and widespread dissemination of virus information so that scientists could start working on vaccines and other research as quickly as possible.

Not so long ago, scientists had to wait to receive the DNA sequence by mail or request it from the investigators of the outbreak. But with GISAID, the entire genome was instantly online for any scientist to access.

H7N9 is named after the two proteins on its viral surface — the hemagglutinin, or HA, and neuraminidase, NA. There are at least 17 types of HAs and 10 NAs wafting around in various pairings, but the H7N9 combination has never before been seen in humans, meaning we likely have no immunity to it.

For the vaccine seed strain, Webby honed in on the gene snippets coding the HA and NA proteins — the main ingredients in the vaccine recipe for triggering an immune response to attack the virus.

To create the seed strain, Webby needed the genes that would code for the HA and NA — so he had them synthesized. Webby sent the DNA sequences to a biotech company and, after a couple of days and thousands of dollars, the genes arrived.

Meanwhile, across the street from Webby's lab, preparations were being made at St. Jude's Good Manufacturing Practice facility, where Jim Allay works. Among the first tasks at hand was to order the eggs.

Since the 1940s, flu vaccines have been produced in fertilized eggs. Today, new technologies are coming into play but flu vaccine manufacturers are still stuck on eggs — they are cheap, time proven and provide a naturally sterile environment for the virus to grow.

But, of course, eggs come with their own unique production problems — the process is slow, the eggs can spoil, and the amount of virus grown is inconsistent. And if a pandemic were to strike in the middle of summer, then you've got the rooster's virility to worry about.

“In the heat, fertility goes down. The roosters don't do so well,” explains Dr. Earl Brown, an influenza virologist at the University of Ottawa, who was not involved with the seed strain. “In the winter, we'll have 80 to 90 per cent embryos; (in the summer), it (may) go down to 50 per cent.”

And the eggs can't just be bought from your local farmer. For the H7N9 seed strain, St. Jude purchased eggs (at $4.50 apiece) from a company that raises chickens specifically for vaccine production — meaning the hens and roosters live in a completely sterile environment.

After the eggs were delivered from Chicago by courier, they went into an incubator that mimics their natural environment: warm and humid. The eggs were also periodically shifted, just as a mother hen would do.

With the HA and NA genes in hand, Allay got to work growing the seed strain. Flu viruses are made up of eight gene segments; for the seed strain, two came from the synthetic HA and NA. The other six were donated by a strain called PR8, an H1N1 virus that scientists isolated in 1934 in Puerto Rico. It continues to be used for vaccines today because PR8 doesn't make people sick and it grows well in eggs.

But things got off to a less-than-stellar start for the H7N9 seed strain. The first step was to grow the virus in a small batch of just 15 eggs — but only three eggs grew any virus. “That's on the lower side of normal,” Allay admits.

Next, the virus was diluted and cultured in kidney cells from a chicken embryo and African green monkey — a combination of cell types that seems to work best for growing H7 viruses. This step was performed twice and ensures that the final vaccine strain is as pure as possible.

Once the embryos were 10 days old, Allay and his colleagues took them into a Biosafety Level 3 laboratory — the second highest biocontainment level. After removing the virus from the cell cultures, they drilled tiny holes into the eggshells and injected the virus. The holes were then sealed with a dot of paraffin wax and the eggs went back into the incubator. They stayed there for two days, until Sunday night, when they were moved into a refrigerator — thus killing the embryos but preserving any virus that might have grown.

But did any virus grow? That was the million-dollar question on Monday morning. The minute Allay woke up, his thoughts immediately turned to those eggs and what he might find inside them.

What Allay hoped to find was a virus that grew well. The batch would need a high titre, which measures whether enough virus had grown to produce a viable seed strain. The titre test took about 20 minutes — what Allay describes as “some of the longest 20 minutes around.”

“It's a little bit of holding your breath time until that titre comes,” he said.

When the test results were ready, there was one surprise: one batch didn't produce any virus at all. But the other batches — 120 eggs between them — did.

The titres were on the low side, however — only 128 “HA units,” Allay says. A good growing virus, by comparison, would have HA units three or four times greater, he explained.

“This is not one of the greatest growing viruses,” he says. “It's not out of this world — but it's enough.”

The final steps for this seed strain are to ensure it doesn't cause harm by testing it in ferrets, the best laboratory replacement for humans when it comes to flu. The strain also undergoes a slew of quality control tests — in 2006, a single bacterium somehow got into another seed strain and Allay and his team had to spend five months investigating the contamination before they could resume their work.

If all goes well with this seed strain, it will be offered to drug manufacturers, perhaps moving onto clinical testing in humans. The United States might also choose to add it to the national stockpile of pandemic vaccines.

But the seed strain's disappointing titre hints at future problems with the vaccine. Flu experts have already predicted that an H7N9 vaccine will likely be difficult to make — past efforts to develop vaccines for other H7 viruses have been “abysmal,” Webby says. The H7 protein doesn't seem to trigger a good immune response.

This suggests that an H7N9 vaccine could require two vaccine doses and maybe even an adjuvant, a substance that can be added to the virus to give the immune system an extra nudge. (Adjuvants also carry extra controversy; recent studies have suggested that an adjuvant in the 2009 H1N1 pandemic vaccine may be linked to increasing rates of narcolepsy in children).

All of which will only gunk up the vaccine production machinery — which is already slow enough, says flu expert Michael Osterholm with the University of Minnesota.

If an H7N9 pandemic were to erupt tomorrow, vaccines probably wouldn't be available for at least four months — and that's assuming everything goes well, Osterholm recently wrote in an editorial in the Journal of American Medical Association.

“Even with recent additional vaccine manufacturing capacity ... the global public health community remains woefully underprepared for an effective vaccine response to a pandemic,” he and his co-authors wrote.

Back in his office in Memphis, Webby admits that potential issues with the H7N9 vaccine lurk “in the corner of most of our minds.” But if a pandemic becomes imminent, “then we can't make vaccine without these guys,” he says. “(Making a seed strain) is really a no-brainer.”

For now, Webby is focused on producing the best possible seed strain, a task that has consumed his days over the past two months.

In his office, papers are stacked on the floor and his window facing the Mississippi River is covered with H7N9-related notes written in blue and red marker.

But all of this work, he says, is for a vaccine he hopes will never see the light of day.

“I would much prefer to be making these things and they are never ever used,” Webby says. “Because if they were needed, then that means this virus has gone pandemic.”

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